T lymphocyte Migration occurs during homing to lymphoid organs, exit from the vasculature and entry into peripheral tissues. This process involves the adhesive interaction of the T-cell surface with other cells. To examine this phenomenon in vitro human T lymphocytes are isolated, cultured, and placed on tissue culture plates coated with the adhesive protein.
ICAM one and chemokine SDF one. Images of T lymphocyte migration can then be acquired and analyzed. Hi, I'm Craig LeFort and the laboratory of Minsu Kim in the Center for Vaccine Biology and Immunology at the University of Rochester.
Today we'll show you a procedure for the isolation and culture of human teen lymphocytes, an analysis of their migration in vitro. We use this assay in our laboratory to study the role of integrins and T-cell migration. The major integrin in T-cell is lymphocyte function associated antigen one or LFA one.
The specific ligands for LFA one are the icas, such as ICAM one. In addition, a kin signal is required for T-cell migration to provide both a directionality signal as well as to activate integrins. In our assay, we use human ICAM one and CX CL 12 or SDF one as substrates for T-cell migration.
So let's get started. After obtaining human blood from a healthy donor, allow the blood to cool to room temperature. This takes about 30 minutes.
Once the blood is cooled, gently pipette three milliliters of room temperature, polymorph density gradient media into an eight milliliter round bottom polystyrene tube. Then gently add three milliliters of whole blood on top. It is important to avoid any mixing.
Place the tubes in a centrifuge and spin it 500 G for 45 minutes. At room temperature following the centrifugation, the peripheral blood mononuclear cells or P BMCs have been separated from the other blood components. The PBMC layer appears as the first cloudy band from the top.
Under sterile conditions, carefully remove and discard the clear yellow colored upper phase. Then use a P 1000 micro pipette to transfer the PBMC layer to a new conical tube. Wash the PBMC twice with PBS centrifuging cells at 500 G for five minutes.
Each time the supernatant will be somewhat cloudy After each wash. Re suspend the cells in 20 milliliters of RPMI 1640 media containing 10%FBS, 1%penicillin streptomycin, and one microgram per milliliter. Phyto hemagglutinin or PHA incubation in the presence of PHA induces T lymphocyte activation and expansion.
Using a pipette transfer the pbmc to a T 75 culture flask. Next incubated 37 degrees Celsius and 5%carbon dioxide for one to 24 hours. This step allows monocytes, which will adhere to the flask surface to be separated from the lymphocytes that remain in suspension.
Following the incubation, carefully remove all of the media which contains primarily lymphocytes and transfer it to a 15 milliliter conical tube. Centrifuge at 500 G for five minutes. Reese has been the cell pellet in RPMI 1640 and transfer the cells to a new T 75 flaz containing 25 milliliters of RPMI 1640 with FBS, penicillin, streptomycin, and PHA incubated 37 degrees Celsius.
After 24 hours of growth, it may be necessary to add 15 to 20 milliliters of fresh media and transfer to a larger T 1 75 flask. Continue incubating for three days or two days. If the initial incubation of PBMC was overnight after three days, use a pipette to remove the medium, which will contain suspended lymphocytes and transfer it to a 50 milliliter conical tube centrifuge at 500 G for five minutes.
Reese has been the cell pellet and transferred the cells to a new T 75 containing 25 milliliters of RPMI 1640. With FBS, penicillin, streptomycin and human IL two or IL 15 place the cells in the incubator. If starting a T 75 flask, the culture will need to be expanded and transferred to a T 1 75 flask.
After one to two days grow lymphocytes for a total of four to seven days. One day before the migration assay coat a glass bottom 0.17 millimeter dish with 20 micrograms per milliliter of protein A or G in PBS incubate overnight at four degrees Celsius the next day. Wash the dish extensively with PBS, then add ICAM one FC and human SD F1 in PBS solution to the dish.
Incubate for four hours at room temperature to immobilize the molecules. Determine the density of the cultured cells using a hemo cytometer. Then wash the lymphocytes twice with PBS and resuspend them in one milliliter of L 15 media containing D glucose following the incubation, wash the coated dish with ICAM one FC and SDF one extensively with PBS transfer the T lymphocytes in one milliliter of media to the dish, approximately two to five times 10 to the fifth cell should be used per dish to achieve a cell density appropriate for migration analysis.
To obtain images of cell migration, place the dish on the microscope stage in a temperature controlled environment such as a heated chamber at 37 degrees Celsius. Open the NIS elements software in the image settings menu. Choose two by two binning as the mode for both live imaging and image capture.
Go to the applications menu and choose, define, run, experiment. Choose the length of time between images and the total time. For the image capture sequence, press the run button to begin image acquisition following the acquisition.
Migration parameters such as velocity, path length and displacement can be quantified using a software package such as Image J Auto Quant or veloc. To generate a spider web plot, collect the XY coordinates for each cell and time point and project them onto a graph with a common starting point for each cell at the origin. The T lymphocytes shown here were cultured plated on ICAM and SDF one and imaged every 10 seconds for 30 minutes.
This movie shows the random migration of T lymphocytes at a velocity of approximately 15 microns per minute using the XYT coordinates for each cell. 15 cells were chosen at random and plotted with a common starting point at the origin. Comparing spiderweb plots gives a quick visual depiction of differences in migration between different experimental conditions.
Plots for T lymphocyte migration under control conditions are shown on the left while plots for T lymphocyte migration in the presence of an anti LFA one ligand blocking antibody are shown on the right. These data demonstrate that T lymphocytes utilize integrin LFA one to migrate on an ICAM one substrate. We've just shown you how to do a migration assay using cultured human T lymphocytes during the procedure.
It's, it's important to remember to add an appropriate number of cells to the ICAM one coated dish so that cell tracking and analysis of migration are simplified. So that's it. Thanks for watching and good luck with your experiments.
